This invention relates to lipid-binding proteins such as defatted albumin, especially as a spray-dried product, and to their therapeutic and diagnostic use. More particularly, the invention relates to microparticles that can be used as carriers in therapy, e.g. gene therapy, and to the combination of carrier and therapeutic agent.
Human serum albumin (HSA) is a protein whose production in the form of microparticles having a size suitable for use in therapy by parenteral administration or by inhalation, alone or as a carrier for an active agent, e.g. in a metered dose inhaler, is disclosed in WO-A-9609814 and in WO-A-9618388. The HSA may be used as such or as a carrier for a desired active agent, since appropriate spray-drying conditions do not denature the protein or essentially reduce the existence of groups available for binding.
As described in WO-A-9218164, albumin microparticles may be produced in soluble form and then stabilised, for use as diagnostic agents. WO-A-9618388 discloses that such products can be conjugated to therapeutic agents. WO-9609814 discloses that the soluble microparticles are not denaturated, and therefore retain therapeutic utility.
HSA is known to bind a wide range of ligands including drugs, dyes, toxic compounds (e.g. bilirubin) and hydrophobic molecules such as fatty acids. Fatty acid transport is possibly one of the most important functions of HSA. A loading of up to two molecules of lipid is considered normal with a high turnover rate. HSA has the ability to bind fatty acids with high affinity, and this appears to increase with the increasing length of the fatty acid chain. Very high affinities (Ka=107-109) have been reported for long chain fatty acids. HSA is capable of binding up to 6-7 molecules of fatty acid/molecules of protein with moderate to high affinities. Low affinity binding of fatty acids, in numbers as high as 60 molecules of fatty acids/HSA molecule, has been reported.
Fatty acids give added stability to the HSA molecule, and the conformation and subsequent ability to bind ligands are affected by the amount and type of fatty acid bound. Fatty acids also protect against thermal denaturation and the ability of HSA to recover from thermal shock. Long chain fatty acids appear to be better than shorter chain fatty acids. See Brown and Shockley (1982), in xe2x80x9cLipid Protein Interactionsxe2x80x9d, Ed. P. C. Jost and O. H. Griffith, vol 1, pp 25-68, John Wiley, NY; Kragh-Hanson (1990), Danish Medical Bulletin 37:57-84; and Carter and Ho (1994), Advances In Protein Chemistry 45:153-203.
It has now been discovered that lipid-bound proteins such as albumin may associate only weakly with DNA, but that lipid-binding proteins such as defatted albumin, provided as microparticles, have surprisingly enhanced association with DNA. Without wishing to be bound by theory, we note that short-chain fatty acids (e.g. octanoate) are not removed by long-chain fatty acids, suggesting that higher affinity short-chain sites occupy the lower affinity long-chain sites on, say, HSA. Defatting of HSA may therefore make available some or all of the 6-7 high affinity long-chain fatty acid sites and further high affinity short-chain sites.
The ability of lipid-binding proteins such as defatted HSA to associate with DNA may be further enhanced by the addition of a cationic molecule (DNA being anionic). It is a feature of this invention that defatted albumins can be loaded with a wide range of fatty acids, e.g. DC-Chol, or other ligands which would otherwise be inhibited by the presence of the naturally-bound fatty acids. The removal of fatty acids liberates not only these sites but also makes available binding sites for aspirin or other drugs. The removal of a mixed population of fatty acids (and probably other ligands) allows the reloading of specific fatty acids, drugs or intermediate ligands to assist in the stability, conformational structure and/or binding capabilities of microparticles of defatted albumin. The reloading of, for example, aminocaprylic acid may be achieved prior to or after spray-drying of defatted albumin, to give a cationic capsule for DNA binding and parenteral delivery. The microparticles may also be loaded, at the time of administration, with a drug-bound fatty acid (ligand) complex, potentially improving usage, storage, stability and applications.
Further, microparticles of the invention having a modified, predetermined fatty acid profile may be used as enhanced ultrasound contrast agents.
Microparticles according to the invention are obtainable by spray-drying. Suitable conditions are described in WO-A-9218164, WO-A-9609814 and WO-A-9618388. These publications also describe relevant parameters of the microparticles, as regards formulation, size, size distribution etc. These parameters are also preferred for microparticles of this invention. Size and size distribution may be less critical than has been described in the given particles for, say, administration to the alveoli. Microparticles of this invention may be nanoparticles or larger, e.g. up to 50 xcexcm in diameter.
The microparticles may comprise additional components adapted for a particular use. For example, lipid may enhance cell membrane interaction and thus enhance uptake. The lipid itself or any additional component may be introduced by co-spray-drying or by modification of the formed microparticles, before or after stabilisation.
An additional component may be introduced by co-spray-drying or by modification of the formed microparticles, before or after stabilisation.
For use in this invention, a lipid-bound protein such as albumin is defatted. This may be achieved, i.e. fatty acids may be removed, by using acidified activated charcoal, as described by Chen, J. Biol. Chem. 242:173-181 (1967). The charcoal should be washed. Alternatively, solvent extraction may be used.
An albumin is the preferred lipid-binding protein for use in this invention, e.g. in soluble or microparticulate form. The albumin may be naturally-occurring or recombinant. For the purpose of illustration only, the invention will be described below with reference to HSA.
Clinical grade HSA is normally formulated with octanoate (in the presence or absence of tryptophanamide). After removal of this, and defatting, a cationic version of this lipid (e.g. aminocaprylic or aminocaproic acid) can be bound to HSA or HSA microcapsules. Other cationic lipids can also be loaded, pre- or post-spray-drying, producing a vehicle for parenteral delivery of non-viral gene vectors. The utility of albumin microparticles, as a vehicle for gene therapy, and suitable constituents and conditions, are disclosed in PCT/GB97/00953, the contents of which are incorporated herein by reference.
Cationic and/or anionic lipids or ligands that bind to free fatty acid-binding sites on HSA can be used to modify the charge and hydrophilicity or hydrophobicity of the microcapsule. This may be advantageous for the targeting of microcapsules for delivery of cytotoxic and other drugs to specific organs such as lung, liver, spleen etc. For example, by appropriate charge modification, a product of this invention may be adapted to bypass the liver and be transported to the lung.
The reloading of, for example, long-chain fatty acids may lead to an altered but more stable configuration of the protein. The molecules may be modified accordingly, to produce microcapsules with modified shell structure relative to the stabilised/cross-linked products described in WO-A-9218164 (available from Andaris Limited under the trade name Quantison), for improved drug delivery and also for better echogenicity for imaging.
In a therapeutic product of the invention, a therapeutic agent may be complexed with or loaded directly onto defatted HSA microparticles. Alternatively, a fatty acid-agent complex may be formed, e.g. by covalent binding, and used to reload the defatted microparticles.
The lipid that is loaded may itself be a therapeutic agent. For example, the invention is of utility as a vaccine delivery system, e.g. using a lipopeptide. The inclusion of a polycationic or polyanionic tail on the end of a peptide allows the loading of microcapsules with proteinaceous material, and thus provides a means of delivering antigenic peptides. A suitable lipopeptide is described by Allsopp et al, Eur. J. Immunol. (1996) 26:1951-9, where a lipid-tailed peptide induced high levels of cytotoxic T lymphocytes.
The amount of therapeutic agent that is used according to the invention, and its formulation (e.g. with a suitable diluent or carrier) and administration, may be conventional. These factors can readily be determined by one of ordinary skill in the art, depending on the nature and degree of the desired effect.